Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

Time course-dependent changes in the transcriptome of human skeletal muscle during recovery from endurance exercise: From inflammation to adaptive remodeling

Re-programming of gene expression is fundamental for skeletal muscle adaptations in response to endurance exercise. This study investigated the time-course dependent changes in the muscular transcriptome following an endurance exercise trial consisting of 1 h of intense cycling immediately followed by 1 h of intense running. Skeletal muscle samples were taken at baseline, 3 h, 48 h, and 96 h post-exercise from eight healthy, endurance-trained, male individuals. RNA was extracted from muscle. Differential gene expression was evaluated using Illumina microarrays and validated with qPCR. Gene set enrichment analysis identified enriched molecular signatures chosen from the Molecular Signatures Database. Three h post-exercise, 102 gene sets were up-regulated [family wise error rate (FWER), P < 0.05]; including groups of genes related with leukocyte migration, immune and chaperone activation, and cyclic AMP responsive element binding protein (CREB) 1-signaling. Forty-eight h post-exercise, among 19 enriched gene sets (FWER, P < 0.05), two gene sets related to actin cytoskeleton remodeling were up-regulated. Ninety-six h post-exercise, 83 gene sets were enriched (FWER, P < 0.05), 80 of which were up-regulated; including gene groups related to chemokine signaling, cell stress management, and extracellular matrix remodeling. These data provide comprehensive insights into the molecular pathways involved in acute stress, recovery, and adaptive muscular responses to endurance exercise. The novel 96 h post-exercise transcriptome indicates substantial transcriptional activity, potentially associated with the prolonged presence of leukocytes in the muscles. This suggests that muscular recovery, from a transcriptional perspective, is incomplete 96 h after endurance exercise involving muscle damage.

Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment : the M77001 study group

Purpose: This randomized, multicenter trial compared first-line trastuzumab plus docetaxel versus docetaxel alone in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC). Patients and Methods: Patients were randomly assigned to six cycles of docetaxel 100 mg/m 2 every 3 weeks, with or without trastuzumab 4 mg/kg loading dose followed by 2 mg/kg weekly until disease progression. Results: A total of 186 patients received at least one dose of the study drug. Trastuzumab plus docetaxel was significantly superior to docetaxel alone in terms of overall response rate (61% v 34%; P = .0002), overall survival (median, 31.2 v 22.7 months; P = .0325), time to disease progression (median, 11.7 v 6.1 months; P = .0001), time to treatment failure (median, 9.8 v 5.3 months; P = .0001), and duration of response (median, 11.7 v 5.7 months; P = .009). There was little difference in the number and severity of adverse events between the arms. Grade 3 to 4 neutropenia was seen more commonly with the combination (32%) than with docetaxel alone (22%), and there was a slightly higher incidence of febrile neutropenia in the combination arm (23% v 17%). One patient in the combination arm experienced symptomatic heart failure (1%). Another patient experienced symptomatic heart failure 5 months after discontinuation of trastuzumab because of disease progression, while being treated with an investigational anthracycline for 4 months. Conclusion: Trastuzumab combined with docetaxel is superior to docetaxel alone as first-line treatment of patients with HER2-positive MBC in terms of overall survival, response rate, response duration, time to progression, and time to treatment failure, with little additional toxicity. © 2005 by American Society of Clinical Oncology.

A finite volume method with linearisation in time for the solution of advection-reaction-diffusion systems

The numerical solution in one space dimension of advection--reaction--diffusion systems with nonlinear source terms may invoke a high computational cost when the presently available methods are used. Numerous examples of finite volume schemes with high order spatial discretisations together with various techniques for the approximation of the advection term can be found in the literature. Almost all such techniques result in a nonlinear system of equations as a consequence of the finite volume discretisation especially when there are nonlinear source terms in the associated partial differential equation models. This work introduces a new technique that avoids having such nonlinear systems of equations generated by the spatial discretisation process when nonlinear source terms in the model equations can be expanded in positive powers of the dependent function of interest. The basis of this method is a new linearisation technique for the temporal integration of the nonlinear source terms as a supplementation of a more typical finite volume method. The resulting linear system of equations is shown to be both accurate and significantly faster than methods that necessitate the use of solvers for nonlinear system of equations.

Dynamic changes in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers during various murine peripartum states and over time

Mammographic density (MD) is a strong heritable risk factor for breast cancer, and may decrease with increasing parity. However, the biomolecular basis for MD-associated breast cancer remains unclear, and systemic hormonal effects on MD-associated risk is poorly understood. This study assessed the effect of murine peripartum states on high and low MD tissue maintained in a xenograft model of human MD. Method High and low MD human breast tissues were precisely sampled under radiographic guidance from prophylactic mastectomy specimens of women. The high and low MD tissues were maintained in separate vascularised biochambers in nulliparous or pregnant SCID mice for 4 weeks, or mice undergoing postpartum involution or lactation for three additional weeks. High and low MD biochamber material was harvested for histologic and radiographic comparisons during various murine peripartum states. High and low MD biochamber tissues in nulliparous mice were harvested at different timepoints for histologic and radiographic comparisons. Results High MD biochamber tissues had decreased stromal (p = 0.0027), increased adipose (p = 0.0003) and a trend to increased glandular tissue areas (p = 0.076) after murine postpartum involution. Stromal areas decreased (p = 0.042), while glandular (p = 0.001) and adipose areas (p = 0.009) increased in high MD biochamber tissues during lactation. A difference in radiographic density was observed in high (p = 0.0021) or low MD biochamber tissues (p = 0.004) between nulliparous, pregnant and involution groups. No differences in tissue composition were observed in high or low MD biochamber tissues maintained for different durations, although radiographic density increased over time. Conclusion High MD biochamber tissues had measurable histologic changes after postpartum involution or lactation. Alterations in radiographic density occurred in biochamber tissues between different peripartum states and over time. These findings demonstrate the dynamic nature of the human MD xenograft model, providing a platform for studying the biomolecular basis of MD-associated cancer risk. © 2013 Springer Science+Business Media New York.

Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice : real-time PCR quantitation

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial β-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan® real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitate metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 μm segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n = 48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r2 = 0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called 'vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.

Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers

This study investigated the clinicopathologic roles of mammalian target of rapamycin (mTOR) expression and its relationship to carcinogenesis and tumor progression in a colorectal adenoma-adenocarcinoma model. Two colon cancer cell lines with different pathologic stages (SW480 and SW48) and 1 normal colonic epithelial cell line (FHC) were used, in addition to 119 colorectal adenocarcinomas and 32 adenomas. mTOR expression profiles at messenger RNA (mRNA) and protein levels were investigated in the cells and tissues using real-time quantification polymerase chain reaction and immunohistochemistry. The findings were correlated with the clinicopathologic features of the tumors. The colon cell line from stage III cancer (SW48) showed higher expression of mTOR mRNA than that from stage II cancer (SW480). At the tissue level, mTOR showed higher mRNA and protein expression in colorectal carcinoma than in adenoma. The mRNA and protein expression was correlated with each other in approximately one-third of the carcinomas and adenomas. High levels of mTOR mRNA expression were noted more in carcinoma or adenoma arising from the distal portion of the large intestine (P = .025 and .019, respectively). Within the colorectal cancer population, a high level of expression of mTOR mRNA was related to the presence of lymph node metastases (P = .031), advanced pathologic stage (P = .05), and presence of persistent disease or tumor recurrence (P = .035). To conclude, the study has indicated that mTOR is likely to be involved in the development and progression of colorectal cancer and is linked to cancer initiation, invasiveness, and progression.

Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

A polymerase chain reaction-based method for isolating clones from a complimentary DNA library in sheep

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes.

In situ reaction furnace for real-time XRD studies

The new furnace at the Materials Characterization by X-ray Diffraction beamline at Elettra has been designed for powder diffraction measurements at high temperature (up to 1373 K at the present state). Around the measurement region the geometry of the radiative heating element assures a negligible temperature gradient along the capillary and can accommodate either powder samples in capillary or small flat samples. A double capillary holder allows flow-through of gas in the inner sample capillary while the outer one serves as the reaction chamber. The furnace is coupled to a translating curved imaging-plate detector, allowing the collection of diffraction patterns up to 2[theta] [asymptotically equal to] 130°.

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

Human herpesvirus-6 infection in liver transplantation

Rejection and infections are the two most common complications after liver transplantation. Human herpesvirus-6 (HHV-6) belongs to the betaherpesviruses, together with its close relatives cytomegalovirus (CMV) and human herpesvirus-7 (HHV-7). The impact of CMV in liver transplantation is well characterized, but the roles of the other two betaherpesviruses have been acknowledged only recently. Although, HHV-6 reactivation after transplantation is usually asymptomatic, the virus may infect the liver transplant, cause an intragraft lymphocyte dominated inflammatory reaction and graft dysfunction. HHV-6 is also suggested to be associated with liver allograft rejection but the mechanisms are unclear. The aim of this study was to investigate the intragraft immunological processes associated with HHV-6, the involvement of HHV-6 in acute liver failure (ALF) and the hepatic HHV-6 infection of the same patients after transplantation. In addition, the occurrence of HHV-6 and HHV-7 was investigated in liver transplant patients with symptomatic CMV infection. HHV-6 infection of the liver graft was associated with portal lymphocyte infiltration and with a significant increase of adhesion molecules (ICAM-1 and VCAM-1) and the number of cells expressing their ligand molecules (LFA-1, VLA-4) and class II antigens. HHV-6 infection was associated with significant immunological changes, but the immune response was limited to lymphocyte infiltration and the adhesion molecule level. However, one third of these patients developed chronic rejection during the follow-up. Of the patients with ALF of unknown origin, most patients demonstrated HHV-6 antigens in the liver, whereas the opposite was seen in ALF patients with a known disease. After transplantation, HHV-6 recurrence was found in the liver transplant in half of these patients with pre-transplant HHV-6 infection of the liver, whereas no post-transplant HHV-6 infection of the liver was seen in patients without pre-transplant HHV-6. Our studies further demonstrated that both HHV-6 and HHV-7 antigenemia often appeared in association with CMV disease in liver transplant patients. The time-related occurrence of the viruses differed, as HHV-6 appeared early after transplantation and regularly preceded CMV whereas HHV-7 often appeared concurrently with CMV. In conclusion, these results indicate that all three betaherpesviruses are common after liver transplantation, often associated with each other. The immunological events caused by HHV-6 in the liver transplant may be involved in, or trigger mechanisms of allograft rejection. In addition, HHV-6 could be one of the causes of ALF, and pre-transplant HHV-6 infection in ALF patients is a risk factor for post-transplant HHV-6 infection of the graft. These results strongly support the clinical significance of HHV-6 in liver transplantation. Even though the reactivation is usually asymptomatic, in some individuals HHV-6 infection may lead to severe manifestations, such as liver failure or in transplant patients, graft dysfunction and rejection.

Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.